Low Incidence Of GVHD Research Articles

High Disease-Free Survival in Patients with High-Risk MDS Treated with Orca-T

BACKGROUND Allogeneic hematopoietic cell transplant (alloHCT) may be a curative treatment option for patients with myelodysplastic syndrome (MDS). The use of investigational therapies that potentially reduce transplant toxicity could improve outcomes in this patient population with difficult to treat disease. Per CIBMTR, survival after alloHCT for MDS using matched donors is approximately 65% at 1 year and relapse-free survival at 1 year was ~69% in MDS patients in the RICMAC trial who received myeloablative conditioning (MAC). Orca-T is a high-precision cell therapy biologic that includes stem and immune cells, derived from allogeneic donors, that leverages highly purified, polyclonal donor regulatory T cells to control alloreactive immune responses. In this phase 1b study, Orca-T was evaluated in MDS patients who were eligible for transplant per 2017 International Expert Panel recommendations. METHODS As of 03/31/23, 16 patients with intermediate to very high risk (HR) MDS with ≤10% blasts in the bone marrow at the time of transplant had received Orca-T in the phase 1b study. IPSS-M prognostic scores were Low (6%), Moderate Low (6%), Moderate High (19%), High (44%), and Very High (25%). Baseline HCT-CI scores were 0 (n=6), 1 (n=3), 2 (n=2), 3 (n=3), and 4 (n=2). The median f/u was 19.1 months (range 6.3 - 32.2). Patients were aged 43 - 65 (median 59) and 56% female. Donors were 8/8 HLA-matched related (n=7) or unrelated (n=9). Patients received a busulfan-based myeloablative chemotherapy regimen prior to Orca-T, followed by single-agent GvHD prophylaxis with tacrolimus. RESULTS Orca-T was successfully manufactured in a single, centralized GMP manufacturing facility for all patients enrolled, distributed, and infused throughout the U.S. Orca-T was delivered within < 72 hours vein-to-vein (i.e. time between end of donor apheresis to start of recipient's Orca-T infusion). Disease-free survival (RFS) at 12 months was 94% (Figure). At 1 year, no patients had experienced grade ≥ 3 acute GvHD and moderate to severe chronic GvHD had occurred in 2 patients. Modified GRFS (survival free of Grade 3-4 aGvHD, moderate-to-severe cGvHD and relapse) at 12 months was 75%. Non-relapse mortality (NRM) and overall survival (OS) were 0% and 94%, respectively, at 12 months. Additionally, one MDS patient with very poor prognosis, characterized by mutated TP53 and complex karyotype, remains in CR without active signs of acute or cGvHD at 500 days post-alloHCT. CONCLUSIONS Orca-T demonstrated promising results in this HR-MDS patient population, including high RFS, low incidence of GvHD, and very high OS at 1 year. A randomized controlled phase 3 multi-center trial comparing Orca-T to SOC in patients with MDS, AML, and ALL is currently enrolling across the US (NCT05316701).

Post-transplant сyclophosphamide after matched donor hematopoietic stem cell transplantation in children with acute leukemia.

The data on post-transplant cyclophosphamide (PTCy) in pediatric acute leukemia after matched allo-HSCT are limited to case series. The present study aimed to assess the results of PTCy-based GVHD prophylaxis in a large cohort of children with acute leukemia after matched allo-HSCT. A retrospective analysis of 190 pediatric patients with acute leukemia who had a first allograft between 2008 and 2020 from a matched sibling donor (MSD) or matched unrelated donor (MUD) was carried out. In the MSD setting, GVHD prophylaxis consisted of PTCy alone (n=28) for the study group, and calcineurin inhibitor (CNI)±antimetabolite (n=30) for the control group. In MUD setting, most patients in the study group received GVHD prophylaxis with PTCy+CNI+mycophenolate mofetil (n=42, 66.7%) or PTCy+CNI+sirolimus (n=12, 19%). All patients (n=69) in the control group received ATG+CNI+antimetabolite. After MUD allo-HSCT, the incidences of acute GVHD grade III-IV and moderate/severe chronic GVHD were significantly lower in the PTCy group compared to control (6.6%vs. 35.0% and 12.7%vs. 47.1%, respectively, p<.0001). Five-year GVHD-free, relapse-free survival (GRFS) after MUD allo-HSCT was higher in the PTCy group compared to control (35.1%vs. 7.3%, p<.0001). At the same time, there was no significant difference between both groups after MSD allo-HSCT. In pediatric acute leukemia, PTCy-based GVHD prophylaxis for MUD allo-HSCT is a feasible and effective option that results in a low incidence of GVHD. Compared to the ATG-based approach, PTCy provides better control of GVHD in children. In pediatric allo-HSCT from MSD, PTCy demonstrates comparable effectiveness to conventional GVHD prophylaxis.

A Novel Protocol Improves Engraftment in Tαβ + Cell Depleted Haplo-HSCT for Patients with Thalassemia Major

Background: Hematopoietic stem cell transplantation (HSCT) remains a proven curative approach for patients with thalassemia major (TM) over long-term practice. However, achieving successful engraftment and managing graft-versus-host disease (GVHD) continue to pose significant challenges. While low incidence of GVHD has been observed in Tαβ + cell-depleted HSCT (TDH) from haploidentical donors, graft failure (GF) remains a considerable concern, particularly in patients with TM. To enhance the engraftment in TM-TDH, we conducted this prospective/retrospective single-center clinical study. Method: A total of 137 patients diagnosed with TM received TDH at the BMT-center, Taixin Hospital, between March 2021 and April 2023. The median age of the patients was 7 years (ranging from 2 to 19 years), with 89 males and 48 females. The median follow-up time was 408 days (ranging from 99 to 857 days). The conditioning regimen consisted of Thymoglobulin on -day 21, followed by 3-Gy total body irradiation on -day 11. On -day 10, the patients received an infusion of donor lymphocytes (2*10 8/kg), followed by cyclophosphamide (Cy) at a dose of 50mg/kg on days -8, -7, and -2, and 25mg/kg on day -1. Additionally, blood-concentration adjusted busulfan was administered on days -6 to -4, fludarabine at a dose of 40mg/kg on days -6 to -2, and thiotepa at 10mg/kg on day -3. For GVHD prophylaxis, short-term (&amp;lt; 6 months) low-dose tacrolimus or sirolimus with or without mycophenolate mofetil was used. The mean infused CD34+ cells were 31.09 (ranging from 14.25 to 61.12) *10 6/kg, TCRγδ+ cells were 39.49 (ranging from 5.18 to 140.39) *10 6/kg, and NK cells were 109.18 (ranging from 27.52 to 309.41) *10 6/kg, respectively. The targeted infusion of TCRαβ+ cells was aimed at 1.5-2.0*10 5/kg. Results: Out of the 137 patients, 133 achieved successful engraftment (97.1%) with median engraftment times of 16 days (ranging from 9 to 48 days) for ANC to reach 0.5x10 9/L, 11 days (ranging from 1 to 23 days) for HB to reach 80g/L, and 10 days (ranging from 5 to 22 days) for platelets to reach 20x10 9/L, respectively. GF was observed in four patients. Among them, two patients with GF underwent salvage alternative donor second HSCT on +day 19 and +day 32, respectively, and are currently thalassemia-free. One patient received infusion of cryopreserved auto-peripheral blood stem cell and continues to live with TM. Unfortunately, four patients passed away, with one GF patient dying from suspected virus myocarditis on +day 11, one potentially due to virus hepatitis or hepatic GVHD on day +188, and the remaining two from virus bronchiolitis obliterans on +day 211 and +day 254, respectively. The overall survival (OS) and thalassemia-free survival (TFS) rates were 96.5% and 94.3%, respectively. The incidence of GF and transplant-related mortality (TRM) were 2.9% and 3.5%, respectively (Fig.). Acute GVHD of grade I-II and grade III-IV was observed in 10.53% and 9.02% of patients, respectively, while mild and moderate chronic GVHD occurred in 7(5.1%) and 1(0.7%) patient(s), respectively. The incidence of veno-occlusive disease was mild and limited. C onc lusion: The current study demonstrates the efficacy of a novel protocol in enhancing engraftment in TDH for patients with TM and with low incidence of both acute GvHD and chronic GvHD. This approach represents a significant advancement in the field of TM-TDH. Fig.1 Curve of OS, TFS, TRM and Rejection in TM-TDH

Abstract 12 Evaluating the Properties of the Donor to Determine the Quality of Cord Blood

Abstract Introduction Cord blood is a valuable source of hematopoietic stem cells (HSCs) that offer advantages over other sources, but the volume and the absolute number of stem cells in cord blood are limited. Objectives The study aimed to analyze the influence of the factors relating to mothers, babies, and collection on the quality of cord blood units before cryopreservation in Vinmec Tissue Bank, Vietnam. This provides a thorough insight into available predictors that can inform the potential recipients. Methods This study, conducted at Vinmec Tissue Bank in Vietnam between January 2019 and December 2021, collected, processed, and analyzed 2,668 cord blood samples. The study evaluated the impact of donor and collection factors, including maternal age, delivery mode, gestational age, infant weight, gender, blood group, geography, season, collection volume, viability, TNC, and CD34+ cell components on cord blood characteristics. Results Results showed that maternal age, gestational age, delivery mode, and infant weight significantly affected TNC and CD34+ cells, while infant gender only has a significant impact on TNC. Additionally, the collection features such as cord blood volume, geography, the season of delivery, and waiting time associated with cord blood outcomes. Discussion In virtue of its superior matching probability, lower incidence of GVHD, and reduced recurrence rates, cord blood is considered a favorable option for HSC transplantation. However, the limited number of HSCs available from cord blood for bigger patients can be addressed through global cooperation. The possibility of using multiple cord blood units for hematologic cancers has been made feasible through inter-unit HLA matching 29,30. As global cooperation between cord blood banks expands, there is a growing opportunity to find matching samples for recipients. Our large-scale analysis of cord blood throughout Vietnam has highlighted the significance of baby weight and volume in determining cord blood quality. This report represents the first-ever study in Asia to investigate the impact of geography and demography on cord blood. Further evaluation is necessary for a comprehensive understanding of these factors, which would enable more appropriate counseling for patients. Figure 1. Elements that impact the quality of hematopoietic stem cells in cord blood.

GVHD prophylaxis with post-transplant cyclophosphamide results in lower incidence of GVHD and allows faster immunosuppressive treatment reduction compared to antithymocyte globulin in 10/10 HLA-matched unrelated allogeneic hematopoietic cell transplantation.

GVHD prophylaxis with post-transplant cyclophosphamide results in lower incidence of GVHD and allows faster immunosuppressive treatment reduction compared to antithymocyte globulin in 10/10 HLA-matched unrelated allogeneic hematopoietic cell transplantation.

The Clinical Efficacy of Haploidentical Hematopoietic Stem Cell Transplantation by Using Parental Donors in Patients with Thalassemia

To analyze the clinical efficacy of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) by using parental donors on thalassemia patients. The 13 thalassemia patients treated by haplo-HSCT using parental donors in our hospital from July 1, 2016, to July 1, 2020 were retrospectively reviewed. Hematopoiesis reconstitution, the incidence of GVHD, infections and the long-term survival of the patients were analyzed. Twelve of the 13 patients were successfully implanted, the success rate of implantation was 92.3%. The median time of neutrophil and platelet engraftment was 12.5 days (range, 9-22 days) and 21 days (range,12-34 days), respectively. One patient achieved primary graft failure. Three (25%) patients developed to acute GVHD (aGVHD) and achieved complete remission after treatment. Chronic GVHD developed in three (25%) patients, one of them was extensive and under treatment, while one patient developed to severe bacterial infection (7.7%). CMV viremia was diagnosed in two patients (15.4%). There were no patients developed to CMV disease. Three (23.1%) patients achieved EB viremia after transplantation, one of them developed to EBV-related lymphocytic proliferative disease, while there were no patients showed invasive fungal infection. At the last follow-up, all patients survived, twelve of them were free from transfusion dependency. There were no transplant-related deaths. Projected overall and thalassemia-free survival at three years was 100% and 92.3%, respectively. The transplant protocol of haplo-HSCT by using parental donors in patients with thalassemia has reliable source of donors, high incidence of successful implantation and low incidence of GVHD, which can be used as an effective way to increase the source of donors in children with thalassemia.

TCRαβ/CD19 depleted HSCT from an HLA-haploidentical relative to treat children with different nonmalignant disorders

AbstractSeveral nonmalignant disorders (NMDs), either inherited or acquired, can be cured by allogeneic hematopoietic stem cell transplantation (HSCT). Between January 2012 and April 2020, 70 consecutive children affected by primary immunodeficiencies, inherited/acquired bone marrow failure syndromes, red blood cell disorders, or metabolic diseases, lacking a fully matched donor or requiring urgent transplantation underwent TCRαβ/CD19-depleted haploidentical HSCT from an HLA-partially matched relative as part of a prospective study. The median age at transplant was 3.5 years (range 0.3-16.1); the median time from diagnosis to transplant was 10.5 months (2.7 for SCID patients). Primary engraftment was obtained in 51 patients, while 19 and 2 patients experienced either primary or secondary graft failure (GF), the overall incidence of this complication being 30.4%. Most GFs were observed in children with disease at risk for this complication (eg, aplastic anemia, thalassemia). All but 5 patients experiencing GF were successfully retransplanted. Six patients died of infectious complications (4 had active/recent infections at the time of HSCT), the cumulative incidence of transplant-related mortality (TRM) being 8.5%. Cumulative incidence of grade 1-2 acute GVHD was 14.4% (no patient developed grade 3-4 acute GVHD). Only one patient at risk developed mild chronic GVHD. With a median follow-up of 3.5 years, the 5-year probability of overall and disease-free survival was 91.4% and 86.8%, respectively. In conclusion, TCRαβ/CD19-depleted haploidentical HSCT from an HLA-partially matched relative is confirmed to be an effective treatment of children with NMDs. Prompt donor availability, low incidence of GVHD, and TRM make this strategy an attractive option in NMDs patients. The study is registered at ClinicalTrial.gov as NCT01810120.

Gvhd after PBSC-Haploidentical Stem Cell Transplantation (Haplo-SCT) with Post Transplantation-Cyclophosphamide (PT-Cy)

T-cell replete Haplo-SCT using PT-Cy as GVHD part of prophylaxis has become a valid option to perform allogeneic transplantation in patients without an available HLA-matched donor. The absence of additional ex vivo procedure has participated to its increasing use in the field of transplantation. Because of HLA disparity, bone marrow (BM) was initially chosen as stem cell source, based on previous observations of lower incidences of GVHD after HLA-matched donor transplantation, when using BM graft as compared with G-CSF mobilized peripheral blood stem cells (PBSC). In contrast, use of PBSC may improve engraftment and disease control favoring larger adoption of the procedure. However, the use of PBSC graft for Haplo-SCT remains controversial. We here report our experience of PBSC Haplo-SCT using PT-Cy for hematological malignancies.We selected patients (pts) with hematological malignancies who underwent Haplo-SCT using PT-Cy at 2 transplant centers from March 2012 to June 2016, using the following criteria: PBSC as graft source; first allogeneic transplantation; PT-Cy as part of GVHD prophylaxis. Patients with refractory acute leukemia who received sequential conditioning regimens were excluded. GVHD prophylaxis included PT-Cy (D+3 and D+4) in association with calcineurin inhibitors and MMF started from D+5.The aim of this retrospective study was to provide a comprehensive description of GVHD following PBSC Haplo-SCT with PT-Cy. Beyond calculation of the cumulative incidence of both acute and chronic GVHD, we precisely describe GVHD features by grading organ involvement and severity. In addition, we assess the prevalence of GVHD and immunosuppressive treatment (IST) at 3, 6, 9, 12, 15, 18, 21 and 24 months after Haplo-SCT in disease free patients, as well as quality of life.We retrospectively analyzed 181 consecutive pts with a median age of 60 years (19-73). Ninety-seven (54%) pts were affected by myeloid diseases, mostly (29%) AML. Disease risk index was high or very high in 47 pts (26%). Non-myeloablative conditioning regimen (low dose TBI + cyclophosphamide and fludarabine) was used in 92 pts (51%), while the remaining patiens received busulfan-based conditioning regimen, with reduced and myeloablative doses in 67 pts (38%) and 21 (12%) pts, respectively.The median CD34+ and CD3+ cells infused were 5.3 x 10^6/kg (range, 1.5-18.1) and 280 (range, 38-704), respectively.Median time to neutrophil recovery (ANC >500 x106/L) was 21 days (range, 17-112) and 30 days (range, 10-394) to platelet recovery (PLT > 20 G x 109/L).Between 24-48 hours after donor stem cells infusion, 88% of recipients experienced fever with chills (median temperature = 40,1°C). No microbiological proof of infection was found in most pts, while bacterial infection was identified in bloodstream culture in 9 % of pts.Fever completely disappeared the day after the last PT-Cy infused dose in 79% of patients.The incidence of grade II-IV and III-IV aGVHD was 23% and 8% at 100 days, respectively. Among patiens who developed aGVHD, 58 (82%) presented with isolated skin involvement, while gut GVHD was responsible for most grade III-IV GVHD (16/20, 80%).The cumulative incidence of cGVHD was 18% at 2 years. According to NIH classification, half of the patients who developed cGVHD presented with mild forms, without needing systemic treatment (mostly skin and mucosa involvement), while severe forms occurred in 3% of GVHD cases.As shown in Figure 1, at 6 months post Haplo-SCT, IST was discontinued in 40% of disease-free patients. In addition, most patients who were disease-free at 2 years were living with no immunosuppressive treatment (98%) and no GVHD (94%).At 2 years, NRM, relapse, PFS, OS and GRFS were 21%, 17%, 62%, 66% and 50%, respectivelyWe conclude that PBSC Haplo-SCT using PT-Cy is a feasible procedure, with low incidences of severe forms of both acute and chronic GVHD. In addition, we pointed out that almost all surviving patients were living without GVHD and IST at 2 years, suggesting a preserved quality of life. Thus, as previously shown using BM as graft source, PT-Cy allows performing T-replete Haplo-SCT with low incidence of GVHD also using PBSC, with very low long term GVHD-related morbidity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparison of Outcomes after Allogeneic Hematopoietic Stem Cell Transplantation of G-CSF-Stimulated Bone Marrow Versus Non-Stimulated Bone Marrow Versus Peripheral Blood

IntroductionThe use of granulocyte colony-stimulated factor (G-CSF) mobilized peripheral blood stem cells (PBSC) has largely replaced harvested bone marrow (BM) for allogeneic stem cell transplantation. Numerous studies have shown faster neutrophil and platelet engraftment with G-CSF mobilized PBSC, leading to shorter hospitalizations; however PBSC transplantations have been associated to a higher incidence of chronic graft-versus host disease (cGVHD).Since the late 1990´s, harvested BM of healthy donors has been mobilized with G-CSF. This method increases the number of progenitor cells (CD34+) harvested, which leads to faster engraftment than with unstimulated BM (UBM).There are no reports comparing the incidence of acute and chronic GVHD with UBM, MBM, and PBSC used to treat diverse benign and malignant hematologic disorders. In this retrospective study, we compare the transplant outcomes of 160 patients from a single institution.MethodsThe charts of patients who received allogeneic HSCT between the February 2011 and September 2016 at our institution were retrospectively reviewed per an IRB approved protocol.Patients were treated according to current guidelines based on their primary diagnosis and functional status. Donors received Filgrastim (10 mcg/kg/day) for 5 doses prior to harvest. All patients received GVHD prophylaxis with methotrexate (on days +1, 3, 6, 11) and tacrolimus (from day -2 until 4 to 6 months after transplantation).Statistical analyses were performed using Minitab 17 Statistical Software 2010. A preliminary analysis was completed using Stepwise Regression/ Analyses of variance to determine any relevant covariates. ANOVA and chi-square tests were used to examine group differences in demographic and clinical characteristics or time to engraftment.ResultsTwenty-nine patients (18.1%) received MBM, 36 received UBM (22.5%), and 95 received PBSC (59.3%). There were no statistically significant differences in patient sex, primary diagnosis, donor characteristics, or disease status between groups. Statically significant differences among groups in patient age were observed (p=0.032, PBSC>UBM>MBM).Engrafment: MBM was found to have a significantly quicker engraftment of the ANC (13 days), when compared to PBSC (17 days) and UBM (19 days; p=0.01). There were no significant differences in time to engraftment of platelets, between groups.Acute GVHD: MBM was found to be associated with a significantly higher incidence of aGVHD (72%; p=0.025) versus PBSC (45%) and UBM (44%). Odds ratio's indicate that it may be up to nine times more likely and eight times more likely for MBM to develop aGVHD, when compared to UBM (95% CI: 1.15, 9.35)Chronic GVHD: Patient's treated with PBSC were found to have a higher incidence of cGVHD, when compared to MBM (45%) and UBM (39%); however these findings were not statistically significant (p=0.145) but they showed a trend to significance.Discussion and conclusionHLA-matched donors as well as related donors have been associated with lower incidence of GVHD. In our study, 100% of MBM were from related donors, 72% of UBM were from unrelated donors, and 53% of the PBSC donors were unrelated. There was a higher incidence of acute and chronic GVHD in MBM, even though they were from HLA-matched related donors; the results were statistically significant for aGVHD and there was a clear trend on cGVHD. Additionally, despite having increased incidence of acute and chronic GVHD, patients who received MBM had better overall survival; this is likely secondary to graft versus leukemia. The main limitations of the study are the sample size and that it is possible that some of the patients have not developed cGVHD yet, therefore it is necessary to obtain data from a larger cohort and with a longer follow up, additionally, since this is a retrospective study access to grade and stage of GVHD is limited for some patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Sustained Donor Chimerism and Rapid Immune Cell Reconstitution with a Low Probability of Gvhd Following Familial Haploidentical (FHI) CD34 Enriched Stem Cell Transplantation with Pbmnc Addback in Patients with High Risk Sickle Cell Disease (SCD) (IND 14359)

Background Allogeneic stem cell transplantation (AlloSCT) from an HLA-matched sibling donor is the only known curative therapy in high-risk SCD patients (Talano/Cairo, EJH, 2015). Unfortunately only about 15% of high-risk SCD patients have an HLA-matched unaffected sibling donor. T cell depletion has been employed to reduce AGVHD. We reported a very low incidence of GVHD in pediatric recipients receiving CD34 enriched HPC products with PBMNC addback from MUD donors (Geyer/Cairo et al, BJH, 2012). Rapid NK cell reconstitution after AlloSCT is associated with a significant improvement in 1yr OS (Pical-Izard, BBMT, 2015). Recently, we reported promising results for high-risk SCD patients at 1 yr follow-up after FHI CD34 enriched/PBMNC with addback with the probability of 1-yr OS 88.2% (Cairo, et al, JAMA Peds, 2019). Objective To investigate donor chimerism, immune reconstitution and the incidence of GVHD in high-risk SCD patients following AlloSCT using FHI CD34 enrichment/PBMNC (2 × 105 CD3/kg) addback. Methods CD34 cells were enriched using the CliniMACS® system, kindly provided by Miltenyi Biotec, with a target dose of 10 × 106 CD34+ cells/kg. PBMNC were added back to the final CD34 product at a dose of 2 × 10*5 CD3/kg. Whole blood and CD71-enriched erythroid lineage chimerism were determined by STR. Immune cell, subset reconstitution, NK function, CD107a and granzyme B were assessed by flow cytometry as previously described (Geyer/Cairo et al. BJH, 2012, Chu/Cairo et al, Can Imm Res, 2015). Results The cumulative incidence of grade II-IV and late AGVHD was 6.2 % and moderate and/or severe CGVHD incidence was 6.7%, respectively (Fig. 1A). There was 100% engraftment of neutrophils and platelets. The median day post-HISCT to neutrophil and platelet engraftment was +9 and +19, respectively. Whole blood donor chimerism (mean±SEM) at 1-yr, 2-ys, and 3-ys post-HISCT was 97±1%, 97±1%, 97±1%, respectively (Fig.1B). Donor chimerism for CD71-enriched erythroid lineage cells (mean±SEM) at 1-yr, 2-yrs, 3-yrs post-HISCT was 97±2%, 98±1%, 98±1%, respectively (Fig.1B). The time to recovery of minimal normal levels for CD3 (800 cells/ul), CD4 (400 cells/ul), CD8 (200 cells/ul), & CD19 cells (200 cells/ul), was approximately 365, 365, 270, and 60 days post-HISCT, respectively (Fig.1C). NK reconstitution was rapid and peaked at d+30 (36±9%) with higher level of activating receptors NKp46, NKG2D & KIR2DS and inhibitory receptors NKG2A, CD94 and KIR2DL2/3. NK cytotoxicity against K562 (E:T = 10:1) peaked at d+30 and d+180 vs pre-transplant (p Conclusion The PBMNC addback facilitated rapid donor chimerism & immune reconstitution with a low probability of Grade II-IV AGVHD. The rapid NK reconstitution may have in part contributed to the excellent 1yr OS in this study (FDA R01FD004090 (MSC)). This approach is comparable to the method of CD3 TCRa/b-CD19 depletion.

Excellent Engraftment with Reduced Treatment Related Mortality for Young Pediatric Patients Using Umbilical Cord Blood Transplantation (UCBT) Conditioned without Serotherapy

Introduction There is no consensus regarding the best donor for children undergoing stem cell transplantation in the absence of a matched related donor (MRD), especially for patients with nonmalignant diseases. The incidence of infections, GvHD, and graft failure remain major problems. Objectives Evaluate outcome for patients undergoing UCBT conditioned without serotherapy, which is as an attractive option for these patients because of immediate availability, lower incidence of GvHD, and high probability of adequate cell dose for young patients. Methods From 2008-2019, we performed UCBT in 84 children, median age 10 mo. (range, 2–108 mo.) with: ALL (13), AML (15), MDS (5), SCID (36), IPEX (2), LAD (1), WAS (1), CGD (1), metabolic disorders (4), HLH (2), and hematologic disorders (4). Eleven patients (30%) with malignancies had minimal residual disease present before UCBT. Five patients with AML had chloromas present prior to UCBT. 26 patients with immune deficiencies had persistent infections at transplant. All patients were enrolled on prospective clinical trials. Pts with AML/ MDS/ or nonmalignant disorders received busulfan, cyclophosphamide, and fludarabine; pts with ALL received TBI (12Gy), cyclophosphamide, and fludarabine. No patient was treated with serotherapy. All cord blood units were matched 5 or 6/6 HLA antigens at A, B and allele at DR. Results Infused cord blood contained median TNC 15 × 107 kg (range, 5.1 – 26.4). All evaluable patients engrafted by day 42. The median time to neutrophil and platelet recovery were 18 days (range, 6-42d) and 35 days (range,22-85d), respectively. All evaluable patients achieved full donor chimerism (defined as > 95% donor cells in peripheral blood by day +42). The overall survival (OS) at 2 years was 75% with a median follow up of 5 years (range: 0.5 – 11yr). The OS for patients with SCID was 92% (33/36) at 2 years with engraftment of all lineages. The cumulative incidence of aGvHD grade II-IV by day 100 was 9% (n=8) with only 3 patients having grade IV. No pt developed cGvHD. Twenty-one patients died, 13/21, 62% died from disease relapse or disease progression. Treatment related mortality occurred for 7 patients; severe GvHD (n=2) and MOF (n=5). No infection related mortality was seen. Conclusion We conclude that lacking a MRD, UCBT following myeloablative conditioning without serotherapy is an excellent curative option in young children. Our experience highlights minimal treatment related mortality, rapid engraftment, resolutions of antecedent infections, and a low incidence of GvHD. These results translate in a better quality of life for these young patients.

Non-Myeloablative Conditioning Regimen before T-Cell Replete Haploidentical Transplantation with Post-Transplant Cyclophosphamide for Advanced Lymphoma

Introduction: allogeneic transplantation (allo-HSCT) is a curative treatment for patients with advanced lymphoma. Haploidentical (haplo-SCT) transplantation extended the accessibility to allo-HSCT, overcoming the issue of donor availability. However, alternative donor allo-HSCT is still considered at higher risk of non-relapse mortality due to the HLA disparity and thus an anticipated higher incidence of GVHD. In this context, the use of a non myeloablative conditioning (NMAC) regimen combined with post transplantation cyclophosphamide (PT-Cy) based GVHD prophylaxis may reduce procedure related toxicity. The aim was to evaluate the toxicity and efficacy of haplo-SCT using NMAC with PT-Cy in advanced lymphoma patients. Methods: We here report the retrospective experience of a bicentric transplantation program. We analyzed a cohort of lymphoma patients undergoing Haplo-SCT and homogeneously receiving NMAC and PT-Cy. Inclusion criteria were: 1) first allo-HSCT for advanced lymphoma between 2009 and 2018; 2) haploidentical donor; 3) NMAC (fludarabine cyclophosphamide and 2 gray TBI GVHD prophylaxis consisted of PT-Cy day+3 and +4 , cyclosporine A and MMF starting from day +5. Multivariate analyses included age, disease type (NHL vs HL), HCT-CI (&lt; vs ≥ 3), graft source (PBSC vs BM), disease status at haplo-SCT (CR vs other). Results: One hundred forty seven patients (73 NHL; 74 HL) with a median age of 46 years (range: 19-71) were included. PBSC (peripheral blood stem cell) was used as graft source in 96 patients (65%). Patients received a median number of 3 conventional chemotherapy lines before haplo-SCT (1-8). Sixty-five (44%) had relapse after Auto-HCT. At the time of haplo-SCT, 96 patients (66%) were in complete remission. The cumulative incidences of day+100 grade 2-4 and 3-4 acute GVHD were 30% and 3%, respectively. The cumulative incidences of 2-year chronic and moderate or severe chronic GVHD were 13% and 8%, respectively. With a median follow up of 39 months (6-114), 2-year NRM was 14%, with a trend for higher risk in patients with HCT-CI ≥ 3 (HR 0.39, 95CI [0.15-1.04] p = 0.061) while age was not associated with an increased risk of NRM (HR 1.01, 95CI [0.98-1.05], p = 0.450). Two-year cumulative incidence of relapse (CIR) was 21% and 18% in HL and NHL patients, respectively. Disease status at the time of haplo-SCT was strongly associated with relapse (HR 2.99, 95CI [1.41-6.35], p = 0.004) In HL patients, 2-year PFS, OS and GRFS were 65%, 77% and 57%, respectively, while corresponding values in NHL patients were 65%, 69% and 55%, respectively. Two-year PFS and GRFS were significantly higher in patients who underwent haplo-SCT in CR (PFS: CR vs. no CR: 72% vs. 55%, p=0.045; GRFS: CR vs. no CR: 63% vs. 42%, p=0.010). There was a trend for better 2-year OS in CR (OS: CR vs. no CR: 78% vs. 63%, p=0.063. Conclusion: We confirm the feasibility of haplo-SCT using NMAC and PT-Cy with low incidence of GVHD (notably severe forms) and NRM. In addition, we observed a relatively low incidence of relapse (19%) in this cohort of heavily pretreated patients, underlining a potent graft-versus-lymphoma effect after haplo-SCT, leading to promising survivals, including high rate of GRFS (&gt;50%), suggesting a preserved long term quality of life in survivors. We conclude that NMAC haplo-SCT with PT-Cy should be considered as a valuable curative option for advanced lymphoma patients, with a favorable toxicity profile and promising long term survival. Figure Disclosures Stoppa: celgene: Other: travel fees, lecture fees; takeda: Other: travel fees. Carlo-Stella:MSD: Honoraria; BMS: Honoraria; Janssen: Other: Travel, accommodations; Boehringer Ingelheim: Consultancy; Genenta Science sr: Consultancy; Sanofi: Consultancy, Research Funding; ADC Therapeutics: Consultancy, Other: Travel, accommodations, Research Funding; Novartis: Consultancy, Research Funding; Servier: Consultancy, Honoraria, Other: Travel, accommodations; F. Hoffmann-La Roche Ltd: Honoraria, Other: Travel, accommodations, Research Funding; Rhizen Pharmaceuticals: Research Funding; Celgene: Research Funding; Amgen: Honoraria; Takeda: Other: Travel, accommodations; Janssen Oncology: Honoraria; AstraZeneca: Honoraria. Chabannon:EBMT: Other: Working Party Chair, Board member; Fresenius Kabi: Other: research support; Miltenyi Biotech: Other: research support; Terumo BCT: Other: speaker's fees; Celgene: Other: speaker's fees; Novartis: Other: speaker's fees; Gilead: Other: speaker's fees, hospitalities; Sanofi SA: Other: research support, speaker's fees, hospitalities. Santoro:Takeda: Speakers Bureau; BMS: Speakers Bureau; Roche: Speakers Bureau; Abb-Vie: Speakers Bureau; Amgen: Speakers Bureau; Celgene: Speakers Bureau; Servier: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; AstraZeneca: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Arqule: Consultancy, Speakers Bureau; Lilly: Speakers Bureau; Sandoz: Speakers Bureau; Eisai: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Bayer: Consultancy, Speakers Bureau; MSD: Speakers Bureau; BMS: Consultancy. Blaise:Sanofi: Honoraria; Jazz Pharmaceuticals: Honoraria; Molmed: Consultancy, Honoraria; Pierre Fabre medicaments: Honoraria.

Pre-Engraftment Immune Reactions Has Unique Graft-Versus-Leukemia Effects after Single Cord Blood Transplantation

&lt;Introduction&gt; Cord blood transplantation (CBT) has now become one of the alternative stem cell sources for patients with a wide variety of hematological diseases. Considering that cord blood (CB) grafts contain T cells with an immunologically naïve phenotype, concerns have been raised regarding the capability of CB-derived lymphocytes to mediate sufficient graft-versus-leukemia (GVL) effects. However, the incidence of relapse after CBT is reportedly comparable to that after other stem cell sources. Moreover several recent studies have shown even lower relapse rate in CBT compared to the others, despite a lower incidence of GVHD. We and others have reported a unique clinical manifestation, termed pre-engraftment immune reactions (PIR), that develop early after CBT and before engraftment. The unique immune-mediated manifestation may have the potential to cause GVL effect from very early time point after CBT besides GVHD. &lt;Methods&gt; We conducted a retrospective study of 551 CB recipients to investigate the GVL effect of the PIR after CBT. Patients who underwent single CBT at our institute from 2004-2017 were included. Only those in non-remission status were included. Patients who had a prior history of allogeneic transplant, who had active infections, who had poor PS, and who received ATG or short-term MTX as GVHD prophylaxis were excluded. The diagnosis of PIR was made when patietns showed a high fever (=&gt;38℃) and at least 2 of the following 5 symptoms (skin eruptions, diarrhea, jaundice, weight gain, and peripheral edema) developed six or more days before neutrophil engraftment. Patients who presented with severe organ damage, such as lung, kidney, or liver dysfunction, were classified as severe type. This study was approved by the institutional review board. &lt;Results&gt; The median age was 57 (range, 16-77) years. All patients had hematological malignancies and all were in non-remission status. Sixty-five percent of these patients received MAC regimens, 63% received tacrolimus plus MMF as GVHD prophylaxis, and 86% received a CB unit with 2 or 3 antigen-mismatch, and the median numbers of TNC and CD34-positive cells were 2.59x107/kg and 0.89x105/kg, respectively. The cumulative incidences of total PIR and severe PIR were 69.3% and 20.0%, respectively, with a median onset of 8 days (range, 5-19) post-CBT. Patients who developed PIR were treated with systemic corticosteroids at the discretion of the primary physicians. Younger age (&lt;55 years) or a lower dose of CD34+ cells were identified as significant factors associated with the development of PIR on univariate analysis, and only younger age was significant on multivariate analysis. Patients with PIR (n=382) showed a significantly lower relapse rate than those without PIR (n=169) (27.2% vs 48.8% at 2 years, P&lt;0.01), while there was no major difference in the non relapse mortality (NRM) rate between the two groups (35.7% vs 34.9% at 2 years, P=0.88) (Figure 1A and 1B). Patients with severe PIR (n=110) showed a more pronounced decrease in relapse incidence (12.5% in severe PIR group vs 33.1% in mild PIR group vs 48.8% in PIR (-) group, P&lt;0.01). The cumulative incidence of NRM in the group, however, was extremely high compared to the others (73.3% in severe PIR group vs 20.8% in mild PIR group vs 34.9% in PIR (-) group, P&lt;0.01). Patients with mild PIRs (n=272) showed reductions of both relapse and NRM compared to those without PIRs. The lower incidence of relapse in the PIR group was translated to better 2-year overall survival (OS) compared to those without PIR (41.2% in PIR (+) group vs 31.5% in PIR (-) group, P&lt;0.01) (Figure 1C). Two-year OS of those with mild PIR was better than the other 2 groups (52.0% in mild PIR group vs 31.5% in PIR (-) group vs 13.9% in severe PIR group, P&lt;0.01) (Figure 1D). Patients with severe PIR showed worse OS, mainly due to the higher incidence of NRM. A higher GVL effect was observed in patients with myeloid malignancies than in those with lymphoid malignancies. &lt;Conclusions&gt; Our data clearly showed that development of PIR after single CBT reduces relapse rate without increasing NRM and contributes to superior survival rate in advanced hematological malignanicies. PIR is one of the clinically important factors associated with unique GVL effects after single CBT. Figure 1 Disclosures No relevant conflicts of interest to declare.

TLI in pediatric patients.

Hematopoietic progenitor cell transplantation (HSCT) is a procedure used in different hematological diseases as part of the curative treatment, so the investigators propose a system of conditioning of reduced intensity based on total lymphoid irradiation (TLI) as an alternative to the classic total body irradiation (TBI) followed by haploidentical transplantation in patients compatible with a single HLA haplotype, as an alternative to patients who do not have an HLA compatible donor. A cohort of 25 patients with hematological disease underwent haploidentical HSCT from February 2015 to May 2018, conditioned with TLI from day -10 (2-4days of treatment) followed by thiotepa (5mg/kg/12h) and melphalan (70mg/m2/day) prior to HSCT and prophylaxis with ciclosporin (1.5mg/kg/12h). 2Gy/fraction was administered to complete 8Gy with IMRT and VMAT technique. 12% rejection of the transplant was obtained with acute GVHD < II (48%) and chronic GVHD 12%. No acute toxicity was recorded in irradiated patients and 56% survival of patients at the end of follow-up. Conditioning the haploidentical transplant with TLI, IMRT, and VMAT techniques compared with TBI and RT3D-C techniques is a feasible technique that helps inducing the necessary immunosuppression in patients with a high risk of graft rejection, minimal adverse effects, low incidence of GVHD, and high survival rate.

Abstract 2310: Functional demonstration of CD19 chimeric antigen receptor (CAR) engineered Epstein-Barr virus (EBV) specific T cells: An off-the-shelf, allogeneic CAR T-cell immunotherapy platform

Abstract Chimeric antigen receptor (CAR) T cells have transformed the treatment of advanced B cell malignancies, but broad application has been limited due to technical and operational challenges in the autologous approach. Virus-specific T cells maintain native T cell receptors and have an established clinical profile and potential utility as an off-the-shelf, allogeneic CAR T-cell immunotherapy platform. Tabelecleucel (tab-cel®) is an investigational off-the-shelf, allogeneic T-cell immunotherapy that utilizes endogenous T cell receptors targeting EBV antigens associated with select lymphomas and solid tumors. Tab-cel® functions through endogenous TCRs restricted to EBV antigens and has been shown to be generally well tolerated with low incidence of GvHD and cytokine release syndrome, as well as efficacy in EBV+ post-transplant lymphoproliferative disorders (PTLD). Introduction of CAR transgenes into EBV-specific T cells provides an appealing approach for developing off-the-shelf, allogeneic CAR T immunotherapies. To evaluate feasibility for this platform, we engineered EBV-specific T cells to express second-generation CD19 CARs, utilizing CD28 or 4-1BB co-stimulatory domains. Resulting allo-EBV.CD19.CAR T cells exhibit high expression of both the CD19 CAR and EBV TCR. Additionally, allo-EBV.CD19.CAR T cells demonstrate an enriched central memory phenotype with higher frequency expression of CD62L, CCR7, and CD45RO. Allo-EBV.CD19.CAR T cells also show increased frequency of activation markers CD25 and 4-1BB, and produce activation-associated levels of IFN-γ comparable to EBV-specific T cells. Allo-EBV.CD19.CAR T cells exert potent and specific cytotoxicity against CD19-positive Nalm6 and Raji cells but have limited activity against CD19-negative K562 cells. Both CD19.CAR T and allo-EBV.CD19.CAR T-mediated cytotoxicity displayed rapid and comparable kinetics. Stimulation of allo-EBV.CD19.CAR T cells with either EBV- or CD19-positive targets also induced robust proliferation over several days of antigen challenge. Like unmodified EBV-specific T cells, allo-EBV.CD19.CAR T cells retain the ability to kill B-lymphoblastoid cell lines (BLCL) but spare autologous and allogeneic PHA-blast targets lacking CD19 and EBV antigen expression. This specificity in cytotoxicity is further supported by inflammatory cytokine profiles of effector-target cultures. From these results, allo-EBV.CD19.CAR T cells demonstrate efficient and effective lysis of antigen-positive target cells, while maintaining low allo-cytotoxic potential. These findings establish feasibility for engineering EBV-specific T cells by leveraging next-generation CAR technologies, and support further development as an off-the-shelf, allogeneic CAR-T immunotherapy platform to generate IND clinical candidates. Citation Format: Rhine R. Shen, Christina D. Pham, Michelle Min Wu, Daniel J. Munson, Blake T. Aftab. Functional demonstration of CD19 chimeric antigen receptor (CAR) engineered Epstein-Barr virus (EBV) specific T cells: An off-the-shelf, allogeneic CAR T-cell immunotherapy platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2310.

CD19 chimeric antigen receptor (CAR) engineered epstein-barr virus (EBV) specific T cells – an off-the-shelf, allogeneic CAR T-cell immunotherapy platform

Background & Aim Chimeric antigen receptor (CAR) T cells have demonstrated potent antitumor efficacy in treating a range of B-cell malignancies, yet technical and commercial challenges using an autologous approach hinder widespread development. Virus-specific T cells present a potential strategy towards an off-the-shelf, allogeneic platform due to the volume of supporting clinical data and additional advantages associated with native TCR engagement to latent virus antigens. Tabelecleucel (tab-cel®) is an investigational off-the-shelf, allogeneic T-cell immunotherapy utilizing native TCR specificity against Epstein-Barr virus (EBV) antigens present in lymphomas and other cancers. With low incidence of GvHD and no cytokine release syndrome, tab-cel® has been shown to be efficacious and well tolerated. Transfer of CAR transgenes into EBV-specific T cells represents an attractive approach to generate off-the-shelf, allogeneic CAR T therapies. Methods, Results & Conclusion We have generated EBV-specific T cells engineered with second generation CD19 CARs as tool compositions to evaluate the potential of this off-the-shelf CAR T approach. Allo-EBV.CD19.CAR T cells express high levels of CD19 CAR and maintain native EBV TCR expression. Allo-EBV.CD19 CAR T cells are predominantly central memory and show CD25, 4-1BB, and IFN-γ activation markers. Functional studies also demonstrate potent antitumor activity against CD19-positive NALM/6 and Raji tumor targets, but no activity against CD19-negative K562 cells. Allo-EBV.CD19.CAR T cells exhibit rapid and comparable killing kinetics compared to CD19.CAR T cells. Allo-CAR T cells also demosntarted antigen-specific proliferation against EBV and CD19-positive targets. Like unmodified EBV-specific T cells, allo.EBV.CD19.CAR T cells maintain the ability to kill B-lymphoblastoid cell lines (BLCL) and little reactivity or inflammatory cytokine secretion in response to HLA mismatched and antigen-negative targets. CD19 CAR engineered EBV T cells demonstrate efficient and potent killing of antigen-positive target cells, while exhibiting little alloreactivity. This platform provides a framework from which we are developing ATA3219, a next generation off-the-shelf, CD19-targeted CAR T utilizing a novel optimized stimulatory domain.

Excellent Outcomes for Pediatric Non-Malignant Diseases Using Umbilical Cord Blood Transplantation (UCBT) Conditioned without Serotherapy in the Absence of a Matched Related Donor

Introduction There is no consensus about the best donor for children with non-malignant disorders in the absence of a matched related donor (MRD). Objectives Evaluate UCBT as an attractive option for these patients because of prompt availability, lower risk of GvHD, and increased cell dose at young ages. Methods From 2008-2017, we performed UCBT in 42 children, median age 5 mo. (range, 2–108 mo.) with: SCID (30), IPEX (2), LAD (1), WAS (1), CGD (1), metabolic disorders (3), HLH (1), and other hematologic disorders (3). 26 patients had persistent infections prior to transplant: PJP (5), fungal (1), RSV (4), Parainfluenza 3 (4), VZV (1), Norovirus (1), CMV (4), Rhinovirus (2), bacterial (4); with 9 patients having pneumonia (20%), and 7 (17%) patients requiring mechanical ventilation prior to transplantation. Thirty-four percent of patients were 6/6 HLA antigen matched, and 66% were one HLA antigen mismatched. All patients were enrolled on a prospective clinical trial using fully ablative busulfan, cyclophosphamide, and fludarabine without serotherapy. The median TNC was 15 × 107 kg (range, 5.1 – 26.4). Results The median time to neutrophil and platelet recovery were 18 days (range,6-30d) and 35 days (range,22-85d), respectively. All but one evaluable patient achieved full donor chimerism (defined as > 95% donor cells in peripheral blood by day +42). The overall survival (OS) at 2 years was 90% (95% CI:77-96%) with a median follow up of 4 years (range: 0.5 – 8.5yr). The OS for patients with SCID was 93.3% (28/30) at 2 years with engraftment of all lineages. Four patients died, from severe GvHD (n=2) and MOF (n=2). The cumulative incidence of aGvHD grade II-IV by day 100 was 16% (n=7) with only 2 (IV). No cGvHD has been seen. All but three patients developed engraftment syndrome at a median time of 19 days, (range: 6-46) successfully treated with steroids. All patients with viral infections at the time of transplant cleared infection at a median time of 54 days (range, 44-91). ELISpot analysis after resolution of infections showed T cell responses against the pertinent viruses. The median absolute CD3 (x10^6/L) counts by day 42 and 60 were 361 and 733; respectively. The median absolute CD3CD4 counts by day 42 and 60, were 296, and 506; respectively. The median absolute CD3CD8 absolute count by day 42 and 60, were 71 and 114; respectively. IVIg was discontinued at a median 138 days; (range: 52-769d). Median time to begin immunizations was 11 months, (range: 8-26). All evaluable patients have had correction of their immune or metabolic defect including adequate B cell function in SCID patients. Conclusion We conclude that in the absence of a MRD, UCBT following myeloablative conditioning without serotherapy is an excellent curative option in children with non-malignant disorders, producing rapid engraftment, prompt functional immune-reconstitution, and a low incidence of GvHD.

Intrabone transplant provides full stemness of cord blood stem cells with fast hematopoietic recovery and low GVHD rate: results from a prospective study

Umbilical Cord Blood (UCB) represents a valid option for patients with hematopoietic malignancies lacking an HLA matched donor. To overcome the limitation of the low stem cell dose of UCB, the intrabone (IB) route has been proposed. We report the results of a prospective study on a poor-prognosis cohort of 23 patients receiving intrabone single UCB transplant (Clinicaltrials.gov NCT00886522). Cumulative incidence of hematological recovery at day 90 was 82 ± 9% (ANC > 0.5 × 109/L) and 70 ± 10% (platelet > 50 × 109/L) and correlated with CD34 + cells in the graft. NRM was 20 ± 9%. No severe aGVHD and only one extensive cGVHD occurred, with fast immune reconstitution. To test the hypothesis that the direct IB injection could affect the expression of stem cells regulatory pathways, CD34 + cells from BM aspirates at day + 10, + 20, + 30, processed in hypoxic conditions mimicking the BM-microenvironment (7%pO2), were studied for the expression of c-Mpl, Notch1 and CXCR4. We found that the expression of c-Mpl in CD34 + cells at day + 10 significantly correlated with hematological recovery. In conclusion, IB-UCB transplant success is associated with low incidence of GVHD and high-speed platelet recovery; intrabone route may preserve full hematopoietic stemness by direct delivery of UCB stem cells into the hypoxic HSC niche.

Antagonizing PPARγ Expands Human Hematopoietic Stem and Progenitor Cells By Switching on FBP1-Repressed Glycolysis and Preventing Differentiation

Allogeneic hematopoietic cell transplantation (HCT) is widely used as a life-saving treatment for malignant and non-malignant blood disorders. Hematopoietic stem cells (HSCs) are a major contributing cell population for a successful HCT. While cord blood (CB) is an acceptable source of HSCs for clinical HCTbecause of its many advantages including prompt availability, lower incidence of GvHD and virus infection, CB HCT is usually associated with slower time to engraftment especially in adult patients when compared with other cell sources; this is partly due to limiting numbers of HSCs in single cord units. In order to overcome this limitation, ex vivo expansion of CB HSCs has been evaluated in preclinical and clinical studies for improvement of the clinical efficacy of CB HCT. While a number of different ways have been evaluated to ex-vivo expand human HSCs, little is known about the mechanisms involved, and whether efficient expansion of CB HSCs could be achieved by metabolic reprogramming.In a compound screen for potential candidates which could promote ex vivo expansion of CB HSCs, we found that PPARγ antagonist GW9662 treatment significantly enhanced ex vivo expansion of CB phenotypic HSCs (~5 fold) and progenitor cells (HPCs) (~6.8 fold) in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and cytokines (SCF, FL, TPO) when compared with vehicle control. GW9662 significantly increased numbers of CB colony-forming unit (CFU) granulocyte/macrophage (GM) (~1.8 fold) and granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) (~3.2 fold) progenitors after 4 days ex vivo culture. To assess whether the ex vivo expanded CB HSCs enhanced by the PPARγ antagonist were functional in vivo, we performed both primary and secondary transplantation in immunocompromised NSG mice. Engraftment of CB CD34+ cells in primary recipients was significantly increased (~3 fold) both in bone marrow (BM) and peripheral blood (PB) by the cultured cells treated with GW9662. The percentages of both myeloid and lymphoid lineages were enhanced in BM of primary recipients transplanted with GW9662-treated CB CD34+ cells. We also transplanted CB CD34+ cells transfected with control shRNA or PPAR γ shRNA into NSG mice, and consistently found that both myeloid and lymphoid chimerism was enhanced in BM of recipients which were infused with PPAR γ shRNA transfected-CD34+ cells compared with control shRNA transfected-CD34+ cells. Long term reconstituting and self-renewing capability of GW9662-treated CB CD34+ cells with both enhanced myeloid and lymphoid chimerism, was confirmed in PB and BM in secondary recipients. Limiting dilution analysis was performed to calculate SCID-repopulating cells (SRC), a measure of the number of functional human HSCs. The SRC frequency of GW9662-cultured CB CD34+ cells was 4 fold greater than that of day 0 uncultured CD34+ cells, and 5 fold increased above that of vehicle-treated CD34+ cells with cytokines alone.To gain mechanistic insight into how PPARγ antagonism enhances expansion of human CB HSCs and HPCs, we performed RNA-seq analysis. Antagonizing PPARγ in CB CD34+ cells resulted in downregulation of a number of differentiation associated genes, including CD38, CD1d, HIC1, FAM20C, DUSP4, DHRS3 and ALDH1A2, which suggests that PPARγ antagonist may maintain stemness of CB CD34+ cells partly by preventing differentiation. Of interest, we found that FBP1, encoding fructose 1, 6-bisphosphatase, a negative regulator of glycolysis, was significantly down-regulated by GW9662, which was further confirmed by RT-PCR, western blot and flow cytometry analysis. GW9662 significantly enhanced glucose metabolism in CB HSCs and HPCs without compromising mitochondrial respiration. Enhanced expansion of CB HSCs by antagonizing PPARγ was totally suppressed by removal of glucose or by inhibition of glycolysis. Importantly, suppression of FBP1 greatly promoted glycolysis and ex vivo expansion of long-term repopulating CB HSCs (~3.2 fold). Overexpression of FBP1 significantly suppressed enhancedexpansion and engraftment of CB HSCs by PPARγ antagonist. Our study demonstrates that PPARγ antagonism drives ex vivo expansion of human CB HSCs and HPCs by switching on FBP1 repressed glucose metabolism and by preventing differentiation. This provides new insight into human HSC self-renewal, and suggests a novel and simple means by which metabolic reprogramming may improve the efficacy of CB HCT. DisclosuresNo relevant conflicts of interest to declare.

Viral-specific T-cell transfer from HSCT donor for the treatment of viral infections or diseases after HSCT.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative option for treatment of some malignant and non-malignant hematological diseases. However, post-HSCT patients are severely immunocompromised and susceptible to viral infections, which are a major cause of morbidity and mortality. Although antiviral agents are now available for most types of viral infections, they are not devoid of side effects and their efficacy is limited when there is no concomitant antiviral immune reconstitution. In recent decades, adoptive transfer of viral-specific T cells (VSTs) became an alternative treatment for viral infection after HSCT. However, two major issues are concerned in VST transfer: the risk of GVHD and antiviral efficacy. We report an exhaustive review of the published studies that focus on prophylactic and/or curative therapy by donor VST transfer for post-HSCT common viral infections. A low incidence of GVHD and a good antiviral efficacy was observed after adoptive transfer of VSTs from HSCT donor. Viral-specific T-cell transfer is a promising approach for a broad clinical application. Nevertheless, a randomized controlled study in a large cohort of patients comparing antiviral treatment alone to antiviral treatment combined with VSTs is still needed to demonstrate efficacy and safety.